Measurement of the absolute rate of fucosyl glycoprotein synthesis and assembly into HeLa plasma membranes during the cell cycle is an aim achievable with the available technology, should be complete before the end of the second year, and should yield unambiguous interpretations. M protein, in VSV infected HeLa, is an example of a genuine "membrane" nonglycosylated protein. Its intracellular pool size, flow rates into (and out of) the plasma membrane plus some of its chemical characteristics will be observed. This will aid us in assessing whether M can directly assemble into plasma membrane and perhaps will shed some light on the specificities of that process. This project will benefit from greater knowledge on the composition of the plasma membrane fraction, possible purification of the internal component carrying fucosyl glycoprotein and also on the behaviour of fucosyl glycoprotein. Hence it will be assigned a lower priority and started in the second year. We know very little about the process of fucosyl glycoprotein transport inside HeLa cells and assembly into plasma membrane. This project is a preliminary one aimed at determining possible metabolic requirements, i.e., is it possible to block the process in some specific fashion with, for example, ATP synthesis inhibitors? This project also will benefit from what we learn in the other projects, hence it will also be started later than the others. We will utilize the gas chromatograph we have just obtained to determine mannose and fucose in various cell fractions from HeLa cells. The gamma-32P-ATP-enzymatic assay developed by us (Yurchenco and Atkinson, 1975) will be modified for use with GLC to determine mannosyl glycoprotein flows for comparison with fucosyl glycoprotein flows. The unpublished work in another laboratory indicates that these pathways in HeLa S3 cells are different, and should yield significant information on membrane assembly processes. BIBLIOGRAPHIC REFERENCES: Moyer, S.A., Tsang, J.M., Atkinson, P.H. and Summers, D.F. Studies on the oligosaccharide moieties of the glycoprotein of vesicular stomatitis virus. J. Virol. 18:167-175, 1976. Yurchenco, P.D. and Atkinson, P.H. Kinetics of fucosyl glycoprotein pools in HeLa cells. Biochemistry, in press, 1977.